Method Involving 1-Benzotriazolyl Carbonate Esters of Poly(ethylene glycol)

ABSTRACT

The invention provides a method comprising the steps of providing a poly(ethylene glycol) having one terminal hydroxyl group; reacting the terminal hydroxyl group of the poly(ethylene glycol) with di(1-benzotriazolyl)carbonate to form a 1-benzotriazolylcarbonate ester of the poly(ethylene glycol); reacting the 1-benzotriazolylcarbonate ester of the poly(ethylene glycol) with an amino acid to form an amino acid derivative; and reacting the amino acid derivative with a biologically active agent under conditions to form a polymer-active agent conjugate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/154,917, filed Jun. 7, 2011, now U.S. Pat. No. 8,299,173, which is acontinuation of U.S. application Ser. No. 12/756,905, filed Apr. 8,2010, now U.S. Pat. No. 7,977,427, which is a continuation of U.S.application Ser. No. 12/433,874, filed Apr. 30, 2009, now U.S. Pat. No.7,723,432, which is a continuation of U.S. application Ser. No.12/150,136, filed Apr. 25, 2008, now U.S. Pat. No. 7,544,738, which is acontinuation of U.S. application Ser. No. 11/492,133, filed Jul. 24,2006, now U.S. Pat. No. 7,378,469, which is a continuation of U.S.application Ser. No. 10/727,337, filed Dec. 2, 2003, now U.S. Pat. No.7,101,932, which is a continuation of U.S. application Ser. No.10/068,371, filed Feb. 6, 2002, now U.S. Pat. No. 6,710,125, which is adivisional of U.S. application Ser. No. 09/740,556, filed Dec. 18, 2000,now U.S. Pat. No. 6,376,604, and claims the benefit of priority of U.S.Provisional Application No. 60/171,834, filed Dec. 22, 1999, each ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to activated poly(ethylene glycol) derivativesand methods of preparing such derivatives.

BACKGROUND OF THE INVENTION

Covalent attachment of the hydrophilic polymer, poly(ethylene glycol),abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, tomolecules and surfaces is of considerable utility in biotechnology andmedicine. In its most common form, PEG is a linear polymer terminated ateach end with hydroxyl groups:

HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as “HO-PEG-OH” where it is understood that the“PEG” symbol represents the following structural unit:

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—

where n typically ranges from about 3 to about 4000.

PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in which oneterminus is the relatively inert methoxy group, while the other terminusis a hydroxyl group that is subject to ready chemical modification. Thestructure of mPEG is given below.

CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

-   -   mPEG

Random or block copolymers of ethylene oxide and propylene oxide, shownbelow, are closely related to PEG in their chemistry, and they can besubstituted for PEG in many of its applications:

HO—CH₂CHRO(CH₂CHRO)_(n)CH₂CHR—OH

wherein each R is independently H or CH₃.

PEG is a polymer having the properties of solubility in water and inmany organic solvents, lack of toxicity, and lack of immunogenicity. Oneuse of PEG is to covalently attach the polymer to insoluble molecules tomake the resulting PEG-molecule “conjugate” soluble. For example, it hasbeen shown that the water-insoluble drug paclitaxel, when coupled toPEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336(1995).

To couple PEG to a molecule, such as a protein, it is often necessary to“activate” the PEG by preparing a derivative of the PEG having afunctional group at a terminus thereof. The functional group can reactwith certain moieties on the protein, such as an amino group, thusforming a PEG-protein conjugate.

In U.S. Pat. No. 5,650,234, which is incorporated by reference herein inits entirety, a 1-benzotriazolylcarbonate ester of poly(ethylene glycol)is described. The multi-step process described in the '234 patent forforming the 1-benzotriazolylcarbonate ester of PEG includes reaction ofa PEG molecule with the volatile and hazardous compound, phosgene, inorder to form a PEG chloroformate intermediate. The use of phosgene inthe process results in the formation of HCl, which can cause degradationof the PEG backbone. Due to the volatile nature of phosgene, and theresulting safety and quality problems associated with its use, there isa need in the art for a method for preparing 1-benzotriazolylcarbonateesters of PEG without using phosgene.

SUMMARY OF THE INVENTION

The invention provides a method for the preparation of a1-benzotriazolyl-carbonate ester of a water-soluble and non-peptidicpolymer by reacting the polymer with di(1-benzotriazolyl)carbonate(“di-BTC”). Using the invention, the 1-benzotriazolylcarbonate ester canbe formed in a single step and without using phosgene, thereby avoidingthe safety and quality problems associated with that compound.

The method of the invention includes providing a water-soluble andnon-peptidic polymer having at least one terminal hydroxyl group andreacting the terminal hydroxyl group of the water-soluble andnon-peptidic polymer with di(1-benzotriazolyl)carbonate to form the1-benzotriazolylcarbonate ester of the water-soluble and non-peptidicpolymer. Examples of suitable water-soluble and non-peptidic polymersinclude poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide),poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene,polyoxazoline, poly(N-acryloylmorpholine), and copolymers, terpolymers,and mixtures thereof. In one embodiment, the polymer is poly(ethyleneglycol) having an average molecular weight from about 200 Da to about100,000 Da.

The reaction step can be conducted in the presence of an organic solventand a base. Examples of suitable organic solvents include methylenechloride, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide,dimethyl sulfoxide, and mixtures thereof. The base can be, for example,pyridine, dimethylaminopyridine, quinoline, trialkylamines, and mixturesthereof.

The method of the invention can further include reacting the1-benzotriazolylcarbonate ester of the water-soluble and non-peptidicpolymer with the amino groups of a second polymer having a plurality ofprimary amino groups, such as a protein, poly(ethylene glycol),aminocarbohydrates, or poly(vinylamine), to form a cross-linked polymer.Additionally, the 1-benzotriazolylcarbonate ester can be reacted witheither an amino acid, such as lysine, to form a polymeric amino acidderivative, or a biologically active agent to form a biologically activepolymer conjugate.

DETAILED DESCRIPTION OF THE INVENTION

The terms “functional group”, “active moiety”, “activating group”,“reactive site”, “chemically reactive group” and “chemically reactivemoiety” are used in the art and herein to refer to distinct, definableportions or units of a molecule. The terms are somewhat synonymous inthe chemical arts and are used herein to indicate portions of moleculesthat perform some function or activity and are reactive with othermolecules. The term “active,” when used in conjunction with “functionalgroups”, is intended to include those functional groups that reactreadily with electrophilic or nucleophilic groups on other molecules, incontrast to those groups that require strong catalysts or highlyimpractical reaction conditions in order to react. For example, as wouldbe understood in the art, the term “active ester” would include thoseesters that react readily with nucleophilic groups such as amines.Typically, an active ester will react with an amine in aqueous media ina matter of minutes, whereas certain esters, such as methyl or ethylesters, require a strong catalyst in order to react with a nucleophilicgroup.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. “Hydrolytically stable linkages” meansthat the linkages are substantially stable in water and do not reactwith water at useful pHs, e.g., under physiological conditions for anextended period of time, perhaps even indefinitely. “Hydrolyticallyunstable” or “hydrolytically degradable” linkages means that thelinkages are degradable in water or in aqueous solutions, including forexample, blood. “Enzymatically unstable” or “enzmatically degradable”linkages means that the linkage can be degraded by one or more enzymes.As understood in the art, PEG and related polymers may includedegradable linkages in the polymer backbone or in the linker groupbetween the polymer backbone and one or more of the terminal functionalgroups of the polymer molecule.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalorganism, including but not limited to viruses, bacteria, fungi, plants,animals, and humans. In particular, as used herein, biologically activemolecules include any substance intended for diagnosis, cure mitigation,treatment, or prevention of disease in humans or other animals, or tootherwise enhance physical or mental well-being of humans or animals.Examples of biologically active molecules include, but are not limitedto, peptides, proteins, enzymes, small molecule drugs, dyes, lipids,nucleosides, oligonucleotides, cells, viruses, liposomes, microparticlesand micelles. Classes of biologically active agents that are suitablefor use with the invention include, but are not limited to, antibiotics,fungicides, anti-viral agents, anti-inflammatory agents, anti-tumoragents, cardiovascular agents, anti-anxiety agents, hormones, growthfactors, steroidal agents, and the like.

The invention provides a method for the preparation of a1-benzotriazolylcarbonate ester (also referred to as a BTC ester) of awater-soluble and non-peptidic polymer, wherein a terminal hydroxylgroup of a water-soluble and non-peptidic polymer is reacted withdi(1-benzotriazolyl)carbonate, the structure of which is shown below, toform the 1-benzotriazolylcarbonate ester.

Di(1-benzotriazolyl)carbonate, which should not pose significant safetyor handling problems as a reagent and should not cause degradation ofthe polymer backbone, can be purchased as a 70% (w/w) mixture with1,1,2-trichloroethane from Fluka Chemical Corporation of Milwaukee, Wis.

The polymer backbone of the water-soluble and non-peptidic polymer canbe poly(ethylene glycol) (i.e. PEG). However, it should be understoodthat other related polymers are also suitable for use in the practice ofthis invention and that the use of the term “PEG” or “poly(ethyleneglycol)” is intended to be inclusive and not exclusive in this respect.The term, “PEG”, includes poly(ethylene glycol) in any of its forms,including alkoxy PEG, difunctional PEG, multi-armed PEG, forked PEG,branched PEG, pendent PEG (i.e. PEG or related polymers having one ormore functional groups pendent to the polymer backbone), or PEG withdegradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate an immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEG,having the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 3 to about 2000, is one usefulpolymer in the practice of the invention. PEGs having a molecular weightof from about 200 Da to about 100,000 Da are particularly useful as thepolymer backbone.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(—PEG-OH)_(m) in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

Those of ordinary skill in the art will recognize that the foregoinglist of substantially water soluble and non-peptidic polymer backbonesis by no means exhaustive and is merely illustrative, and that allpolymeric materials having the qualities described above arecontemplated.

For purposes of illustration, a simplified reaction scheme for themethod of the invention is shown below.

wherein BT is

L being the point of bonding to the oxygen atom.

In one embodiment, the reaction between the polymer and diBTC takesplace in an organic solvent and in the presence of a base. Examples ofsuitable organic solvents include methylene chloride, chloroform,acetonitrile, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide,and mixtures thereof. Amine bases, such as pyridine,dimethylaminopyridine, quinoline, trialkylamines, includingtriethylamine, and mixtures thereof, are examples of suitable bases. Inone aspect of the invention, the molar ratio ofdi(1-benzotriazolyl)carbonate to the water-soluble and non-peptidicpolymer is about 30:1 or less.

In one embodiment, the water-soluble and non-peptidic polymer has thestructure R′-POLY-OH and the 1-benzotriazolylcarbonate ester of thewater-soluble and non-peptidic polymer has the structure:

wherein POLY is a water-soluble and non-peptidic polymer backbone, suchas PEG, and R′ is a capping group. R′ can be any suitable capping groupknown in the art for polymers of this type. For example, R′ can be arelatively inert capping group, such as an alkoxy group (e.g. methoxy).Alternatively, R′ can be a functional group. Examples of suitablefunctional groups include hydroxyl, protected hydroxyl, active ester,such as N-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, protected amine, protectedhydrazide, thiol, protected thiol, carboxylic acid, protected carboxylicacid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate. The functional group is typicallychosen for attachment to a functional group on a biologically activeagent.

As would be understood in the art, the term “protected” refers to thepresence of a protecting group or moiety that prevents reaction of thechemically reactive functional group under certain reaction conditions.The protecting group will vary depending on the type of chemicallyreactive group being protected. For example, if the chemically reactivegroup is an amine or a hydrazide, the protecting group can be selectedfrom the group of tert-butyloxycarbonyl (t-Boc) and9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is athiol, the protecting group can be orthopyridyldisulfide. If thechemically reactive group is a carboxylic acid, such as butanoic orpropionic acid, or a hydroxyl group, the protecting group can be benzylor an alkyl group such as methyl or ethyl. Other protecting groups knownin the art may also be used in the invention.

In another embodiment, the water-soluble and non-peptidic polymer hasthe structure HO—POLY_(a)-R(POLY_(b)-X)_(q) and the1-benzotriazolylcarbonate ester of the water-soluble and non-peptidicpolymer has the structure

wherein POLY_(a) and POLY_(b) are water-soluble and non-peptidic polymerbackbones, such as PEG, that may be the same or different; R is acentral core molecule, such as glycerol or pentaerythritol; q is aninteger from 2 to about 300; and each X is a capping group. The Xcapping groups may be the same as discussed above for R′.

In another aspect, a difunctional or higher functional BTC ester of thewater-soluble and non-peptidic polymer is reacted with at least twoamino groups of a second polymer having a plurality of primary aminogroups, such as amino PEGs or other multifunctional amine polymers, suchas proteins, aminocarbohydrates, or poly(vinylamine), to formcross-linked polymers. The amine polymer will generally have three ormore available amino groups. Such polymers form hydrogels; that is, theybecome highly hydrated in aqueous media, but do not dissolve. Sincethese hydrogels are commonly biocompatable and may be degradable, manybiomedical applications are possible in the areas of drug delivery,wound covering, and adhesion prevention.

A further embodiment of the invention involves the reaction of BTCesters of water-soluble and non-peptidic polymers with amino acids toform amino acid derivatives. In one embodiment, a PEG-BTC ester isreacted with lysine to form a polymeric lysine derivative. For example,one such lysine derivative is a doubly PEGylated lysine, wherein the twoPEGs are linked to the lysine amines by carbamate bonds, as shown below.

wherein PEG is poly(ethylene glycol) and Z is selected from the groupconsisting of H, N-succinimidyl, or 1-benzotriazolyl.

Such PEG derivatives of lysine are useful as reagents for preparation ofPEG derivatives of proteins. These PEG derivatives often offeradvantages over non-PEGylated proteins, such as longer circulatinglife-times in vivo, reduced rates of proteolysis, and loweredimmunogenicity. In another aspect, PEG BTC derivatives are used directlyin attaching PEG to proteins through carbamate linkages and may offeradvantages similar to those described for the lysine PEG derivatives.

BTC esters of water-soluble and non-peptidic polymers can also bereacted with biologically active agents to form biologically activepolymer conjugates. Examples of biologically active agents includepeptides, proteins, enzymes, small molecule drugs, dyes, lipids,nucleosides, oligonucleotides, cells, viruses, liposomes, microparticlesand micelles.

The invention also includes 1-benzotriazolylcarbonate esters ofwater-soluble and non-peptidic polymers prepared according to theabove-described process. As noted above, it is believed that polymerderivatives prepared according to the invention exhibit higher qualitybecause degradation of the polymer backbone caused by phosgene isavoided. Further, since the method of the invention requires only onestep and fewer reactants, process efficiency is enhanced and cost isreduced.

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention.

EXAMPLES Example 1 Preparation of mPEG₅₀₀₀BTC

A solution of mPEG₅₀₀₀-OH (MW 5000, 15 g, 0.003 moles),di(1-benzotriazolyl)carbonate (4.0 g of 70% mixture, 0.000945 moles),and pyridine (2.2 ml) in acetonitrile (30 ml) was stirred at roomtemperature under nitrogen overnight. The solvent was removed bydistillation, the residue was dissolved in 80 ml of methylene chloride,and the resulting solution was added to 850 ml of ethyl ether. Themixture was cooled to 0-5° C. and the precipitate was collected byfiltration. The precipitation process was then repeated to obtain awhite solid which was dried under vacuum at room temperature to yield13.5 g of product which was shown by ¹H NMR to be 100% substituted. ¹HNMR (dmso d-6): 3.23 ppm, CH₃O; 3.51 ppm, O—CH ₂CH ₂—O; 4.62 ppm, m,mPEG-O—CH ₂—OCO₂—; 7.41-8.21, complex mult., benzotriazole protons.

Example 2 Preparation of mPEG_(20,000)BTC

A solution of mPEG_(20,000)-OH (MW 20,000, 20 g, 0.001 moles),di(1-benzotriazolyl)carbonate (3.4 g of 70% mixture, 0.00803 moles), andpyridine (3.0 ml) in acetonitrile (40 ml) was stirred at roomtemperature under nitrogen overnight. The solvent was removed bydistillation and the residue was dissolved in 80 ml of methylenechloride and the resulting solution was added to 800 ml of ethyl ether.The precipitate was collected by filtration and was dried under vacuumat room temperature to yield 16.8 g of product which was shown by ¹H NMRto be 100% substituted. ¹H NMR (dmso d-6): 3.23 ppm, CH₃O; 3.51 ppm,O—CH ₂CH ₂—O; 4.62 ppm, m, mPEG-O—CH ₂—OCO—; 7.41-8.21, complex mult.,benzotriazole protons.

Example 3 Derivatization of Lysine with mPEG_(20,000) BTC

Lysine.HCl (0.0275 g, 0.000151 moles) was dissolved in 26 ml of 0.1 Mborate buffer and the pH was adjusted to 8.0 with 0.1 M NaOH. To theresulting solution was added mPEG_(20,000) BTC (7.0 g, 0.00350 moles)over 15 minutes and the pH was kept at 8 by addition of 0.1 M NaOH.After stirring the resulting solution for 3 h, 15 g of H₂O and 4 g ofNaCl were added and the pH was adjusted to 3.0 with 10% phosphoric acid.The product was extracted with methylene chloride and the extract driedover MgSO₄. After concentrating the solution to 30 ml, the solution waspoured into 300 ml of ethyl ether and the product collected byfiltration and dried under vacuum at room temperature to yield 5.9 g ofproduct as a white solid. Analysis by gel permeation chromatography(Ultrahydrogel 250, column temperature 75° C., aqueous buffer pH 7.2)showed the product to be a mixture of di-N-PEGylated lysine (MW ˜40 KDa,63.05%), mono-N-PEGylated lysine (MW ˜20 KDa, 36.95%), andmPEG_(20,000).

Example 4 Derivatization of Lysozyme with mPEG₅₀₀₀BTC

To 4 ml of lysozyme solution (3 mg/ml in 50 mM sodium phosphate buffer,pH 7.2) was added 20.3 mg of mPEG₅₀₀₀ BTC (5-fold excess of mPEG5000BTC) and the mixture was continually mixed at room temperature. Analysisby capillary electrophoresis (57 cm×76 um column; 30 mM phosphatebuffer; operating voltage 25 kV) after 4 hours showed that 6.94% ofunreacted lysozyme remained, while 33.99% of mono-PEGylated lysozyme,43.11% di-PEGylated lysozyme, 13.03% tri-PEGylated lysozyme, and 2.92%of tetra-PEGylated lysozyme had formed.

Example 5 PEG_(2KDa)-α-hydroxy-ω-propionic acid, benzyl ester

To a solution of PEG_(2KDa)-α-hydroxy-ω-propionic acid (10 g, 0.0050moles) (Shearwater Corp.) in anhydrous methylene chloride (100 ml),1-hydroxybenzotriazole (0.30 g), 4-(dimethylamino)pyridine (1.0 g),benzyl alcohol (10.8 g, 0.100 moles) and 1,3-dicyclohexylcarbodiimide(1.0 M solution in methylene chloride, 7.5 ml, 0.0075 moles) were added.The reaction mixture was stirred overnight at room temperature underargon. The mixture was then concentrated to about 50 ml, filtered andadded to 800 ml cold diethyl ether. The precipitated product wasfiltered off and dried under reduced pressure. Yield 8.2 g. NMR(d6-DMSO): 2.60 ppm (t, —CH₂—COO—), 3.51 ppm (s, PEG backbone), 4.57 ppm(t, —OH—), 5.11 ppm (s, —CH₂— (benzyl)), 7.36 ppm (m, —C₆H₅ (benzyl)).

Example 6 PEG_(2KDa)-α-benzotriazole carbonate-ω-propionic acid, benzylester

To a solution of PEG_(2KDa)-α-hydroxy-ω-propionic acid, benzyl ester(8.2 g, 0.0025 moles) in acetonitrile (82 ml), pyridine (0.98 ml) anddi(1-benzotriazolyl)carbonate (1.48 g) were added and the reactionmixture was stirred overnight at room temperature under argonatmosphere. The mixture was then filtered and solvent was evaporated todryness. The crude product was dissolved in methylene chloride andprecipitated with isopropyl alcohol. The wet product was dried underreduced pressure. Yield 6.8 g. NMR (d6-DMSO): 2.60 ppm (t, —CH₂—COO—),3.51 ppm (s, PEG backbone), 4.62 ppm (m, —CH₂—O(C═O)—), 5.11 ppm (s,—CH₂-(benzyl)), 7.36 ppm (m, —C₆H₅ (benzyl)), 7.60-8.50 ppm (4 m,aromatic protons of benzotriazole).

1. A method comprising; providing a poly(ethylene glycol)l having oneterminal hydroxyl group; reacting the terminal hydroxyl group of thepoly(ethylene glycol) with di(1-benzotriazolyl)carbonate to form a1-benzotriazolylcarbonate ester of the poly(ethylene glycol); reactingthe 1-benzotriazolylcarbonate ester of the poly(ethylene glycol) with anamino acid to form an amino acid derivative, said amino acid derivativehaving the following structure:

wherein PEG is a poly(ethylene glycol) and Z is selected from the groupconsisting of H, N-succinimidyl, and 1-benzotriazolyl; and reacting theamino acid derivative with a biologically active agent under conditionsto form a polymer-active agent conjugate.
 2. The method of claim 1,wherein each PEG is capped with a capping group.
 3. The method of claim2, wherein the capping group is methoxy.
 4. The method of claim 1,wherein the biologically active agent is a protein.